Androgen receptor complex-associated protein

ABSTRACT

The invention relates to new proteins that bind to and aide the transactivation activity of androgen receptors, and nucleic acids encoding them.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 09/781,693, filed Feb. 12, 2001 and now pending and allowed, whichclaims priority from U.S. Provisional Application No. 60/262,312, filedJan. 17, 2001. The contents of the two parent applications areincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

A variety of genes that are overexpressed in tumor cells relative tohealthy cells have been identified. It is expected that theidentification of such genes will provide drug targets for anti-cancerdrug development and for cancer diagnostics. The number of steroidreceptors (e.g., androgen receptors) in liver tumors cells appears to beincreased relative to their adjacent healthy liver cells.

Steroid hormones generally exert their physiological effects by bindingto their specific nuclear receptors to form complexes that in turn actas transcription factors. The complexes bind to specific nucleotidesequences (steroid responsive elements) in the promoters ofsteroid-responsive genes to facilitate transcription of those genes.

SUMMARY OF THE INVENTION

The invention is based on the discovery of a human protein that isoverexpressed in hepatoma cells relative to normal adjacent tissue inliver cancer patients. It was also discovered that this human proteinbinds to an androgen receptor and moreover augments the ability of theandrogen receptor to transactivate an androgen-responsive gene. Thus,the human protein to which this invention pertains was designatedandrogen receptor complex-associated protein or ARCAP. The full-lengthhuman ARCAP cDNA, with the start and stop codons underlined, is shownbelow.

(SEQ ID NO:3) CCGGCTCAGGCAGAGCCATGTCTCGGGGTGGCTCCTACCCACACCTGTTGTGGGACGTGAGGAAAAGGTCCCTCGGGCTGGAGGACCCGTCCCGGCTGCGGAGTCGCTACCTGGGAAGAAGAGAATTTATCCAAAGATTAAAACTTGAAGCAACCCTTAATGTGCATGATGGTTGTGTTAATACAATCTGTTGGAATGACACTGGAGAATATATTTTATCTGGCTCAGATGACACCAAATTAGTAATTAGTAATCCTTACAGCAGAAAGGTTTTGACAACAATTCGTTCAGGGCACCGAGCAAACATATTTAGTGCAAAGTTCTTACCTTGTACAAATGATAAACAGATTGTATCCTGCTCTGGAGATGGAGTAATATTTTATACCAACGTTGAGCAAGATGCAGAAACCAACAGACAATGCCAATTTACGTGTCATTATGGAACTACTTATGAGATTATGACTGTACCCAATGACCCTTACACTTTTCTCTCTTGTGGTGAAGATGGAACTGTTAGGTGGTTTGATACACGCATCAAAACTAGCTGCACAAAAGAAGATTGTAAAGATGATATTTTAATTAACTGTCGACGTGCTGCCACGTCTGTTGCTATTTGCCCACCAATACCATATTACCTTGCTGTTGGTTGTTCTGACAGCTCAGTACGAATATATGATCGGCGAATGCTGGGCACAAGAGCTACAGGGAATTATGCAGGTCGAGGGACTACTGGAATGGTTGCCCGTTTTATTCCTTCCCATCTTAATAATAAGTCCTGCAGAGTGACATCTCTGTGTTACAGTGAAGATGGTCAAGAGATTCTCGTTAGTTACTCTTCAGATTACATATATCTTTTTGACCCGAAAGATGATACAGCACGAGAACTTAAAACTCCTTCTGCGGAAGAGAGAAGAGAAGAGTTGCGACAACCACCAGTTAAGCGTTTGAGACTTCGTGGTGATTGGTCAGATACTGGACCCAGAGCAAGGCCGGAGAGTGAACGAGAACGAGATGGAGAGCAGAGTCCCAATGTGTCATTGATGCAGAGAATGTCTGATATGTTATCAAGATGGTTTGAAGAAGCAAGTGAGGTTGCACAAAGCAATAGAGGACGAGGAAGATCTCGACCCAGAGGTGGAACAAGTCAATCAGATATTTCAACTCTTCCTACGGTCCCATCAAGTCCTGATTTGGAAGTGAGTGAAACTGCAATGGAAGTAGATACTCCAGCTGAACAATTTCTTCAGCCTTCTACATCCTCTACAATGTCAGCTCAGGCTCATTCGACATCATCTCCCACAGAAAGCCCTCATTCTACTCCTTTGCTATCTTCTCCAGACAGTGAACAAAGGCAGTCTGTTGAGGCATCTGGACACCACACACATCATCAGTCTGATAACAATAATGAAAAGCTGAGCCCCAAACCAGGGACAGGTGAACCAGTTTTAAGTTTGCACTACAGCACAGAAGGAACAACTACAAGCACAATAAAACTGAACTTTACAGATGAATGGAGCAGTATAGCATCAAGTTCTAGAGGAATTGGGAGCCATTGCAAATCTGAGGGTCAGGAGGAATCTTTCGTCCCACAGAGCTCAGTGCAACCACCAGAAGGAGACAGTGAAACAAAAGCTCCTGAAGAATCATCAGAGGATGTGACAAAATATCAGGAAGGAGTATCTGCAGAAAACCCAGTTGAGAACCATATCAATATAACACAATCAGATAAGTTCACAGCCAAGCCATTGGATTCCAACTCAGGAGAAAGAAATGACCTCAATCTTGATCGCTCTTGTGGGGTTCCAGAAGAATCTGCTTCATCTGAAAAAGCCAAGGAACCAGAAACTTCAGATCAGACTAGCACTGAGAGTGCTACCAATGAAAATAACACCAATCCTGAGCCTCAGTTCCAAACAGAAGCCACTGGGCCTTCAGCTCATGAAGAAACATCCACCAGGGACTCTGCTCTTCAGGACACAGATGACAGTGATGATGACCCAGTCCTGATCCCAGGTGCAAGGTATCGAGCAGGACCTGGTGATAGACGCTCTGCTGTTGCCCGTATTCAGGAGTTCTTCAGACGGAGAAAAGAAAGGAAAGAAATGGAAGAATTGGATACTTTGAACATTAGAAGGCCGCTAGTAAAAATGGTTTATAAAGGCCATCGCAACTCCAGGACAATGATAAAAGAAGCCAATTTCTGGGGTGCTAACTTTGTAATGAGTGGTTCTGACTGTGGCCACATTTTCATCTGGGATCGGCACACTGCTGAGCATTTGATGCTTCTGGAAGCTGATAATCATGTGGTAAACTGCCTGCAGCCACATCCGTTTGACCCAATTTTAGCCTCATCTGGCATAGATTATGACATAAAGATCTGGTCACCATTAGAAGAGTCAAGGATTTTTAACCGAAAACTTGCTGATGAAGTTATAACTCGAAACGAACTCATGCTGGAAGAAACTAGAAACACCATTACAGTTCCAGCCTCTTTCATGTTGAGGATGTTGGCTTCACTTAATCATATCCGAGCTGACCGGTTGGAGGGTGACAGATCAGAAGGCTCTGGTCAAGAGAATGAAAATGAGGATGAGGAATAATAAACTCTTTTTGGCAAGCACTTAAATGTTCTGAAATTTGTATAAGACATTTATTATATTTTTTTCTTTACAGAGCTTTAGTGCAATTTTAAGGTTATGGTTTTTGGAGTTTTTCCCTTTTTTTGGGATAACCTAACATTGGTTTGGAATGATTGTGTGCATGAATTTGGGAGATTGTATAAAACAAAACTAGCAGAATGTTTTTAAAACTTTTTGCCGTGTATGAGGAGTGCTAGAAAATGCAAAGTGCAATATTTTCCCTAACCTTCAAATGTGGGAGCTTGGATCAATGTTGAAGAATAATTTTCATCATAGTGAAAATGTTGGTTCAAATAAATTTCTACACTTGCCATTTGCATGTTTGTTGCTTTCTAATTAAGAAACTGGTTGTTTTAAAAAA AAAAAAAAGGAATTCThe nucleotide sequence encoding the human ARCAP protein (i.e., from theATG start codon to the codon immediately before the stop codon in SEQ IDNO:3) is designated SEQ ID NO:1. The ARCAP amino acid sequence encodedby the above cDNA is shown below.

(SEQ ID NO:2) Met ser arg gly gly ser tyr pro his leu leu trp asp valarg lys arg ser leu gly leu glu asp pro ser arg leu arg ser arg tyr leugly arg arg glu phe ile gln arg leu lys leu glu ala thr leu asn val hisasp gly cys val asn thr ile cys trp asn asp thr gly glu tyr ile leu sergly ser asp asp thr lys leu val ile ser asn pro tyr ser arg lys val leuthr thr ile arg ser gly his arg ala asn ile phe ser ala lys phe leu procys thr asn asp lys gln ile val ser cys ser gly asp gly val ile phe tyrthr asn val glu gln asp ala glu thr asn arg gln cys gln phe thr cys histyr gly thr thr tyr glu ile met thr val pro asn asp pro tyr thr phe leuser cys gly glu asp gly thr val arg trp phe asp thr arg ile lys thr sercys thr lys glu asp cys lys asp asp ile leu ile asn cys arg arg ala alathr ser val ala ile cys pro pro ile pro tyr tyr leu ala val gly cys serasp ser ser val arg ile tyr asp arg arg met leu gly thr arg ala thr glyasn tyr ala gly arg gly thr thr gly met val ala arg phe ile pro ser hisleu asn asn lys ser cys arg val thr ser leu cys tyr ser glu asp gly glnglu ile leu val ser tyr ser ser asp tyr ile tyr leu phe asp pro lys aspasp thr ala arg glu leu lys thr pro ser ala glu glu arg arg glu glu leuarg gln pro pro val lys arg leu arg leu arg gly asp trp ser asp thr glypro arg ala arg pro glu ser glu arg glu arg asp gly glu gln ser pro asnval ser leu met gln arg met ser asp met leu ser arg trp phe glu glu alaser glu val ala gln ser asn arg gly arg gly arg ser arg pro arg gly glythr ser gln ser asp ile ser thr leu pro thr val pro ser ser pro asp leuglu val ser glu thr ala met glu val asp thr pro ala glu gln phe leu glnpro ser thr ser ser thr met ser ala gln ala his ser thr ser ser pro thrglu ser pro his ser thr pro leu leu ser ser pro asp ser glu gln arg glnser val glu ala ser gly his his thr his his gln ser asp asn asn asn glulys leu ser pro lys pro gly thr gly glu pro val leu ser leu his tyr serthr glu gly thr thr thr ser thr ile lys leu asn phe thr asp glu trp serser ile ala ser ser ser arg gly ile gly ser his cys lys ser glu gly glnglu glu ser phe val pro gln ser ser val gln pro pro glu gly asp ser gluthr lys ala pro glu glu ser ser glu asp val thr lys tyr gln glu gly valser ala glu asn pro val glu asn his ile asn ile thr gln ser asp lys phethr ala lys pro leu asp ser asn ser gly glu arg asn asp leu asn leu asparg ser cys gly val pro glu glu ser ala ser ser glu lys ala lys glu proglu thr ser asp gln thr ser thr glu ser ala thr asn glu asn asn thr asnpro glu pro gln phe gln thr glu ala thr gly pro ser ala his glu glu thrser thr arg asp ser ala leu gln asp thr asp asp ser asp asp asp pro valleu ile pro gly ala arg tyr arg ala gly pro gly asp arg arg ser ala valala arg ile gln glu phe phe arg arg arg lys glu arg lys glu met glu gluleu asp thr leu asn ile arg arg pro leu val lys met val tyr lys gly hisarg asn ser arg thr met ile lys glu ala asn phe trp gly ala asn phe valmet ser gly ser asp cys gly his ile phe ile trp asp arg his thr ala gluhis leu met leu leu glu ala asp asn his val val asn cys leu gln pro hispro phe asp pro ile leu ala ser ser gly ile asp tyr asp ile lys ile trpser pro leu glu glu ser arg ile phe asn arg lys leu ala asp glu val ilethr arg asn glu leu met leu glu glu thr arg asn thr ile thr val pro alaser phe met leu arg met leu ala ser leu asn his ile arg ala asp arg leuglu gly asp arg ser glu gly ser gly gln glu asn glu asn glu asp glu glu

Accordingly, the invention features a substantially pure polypeptide orprotein including an amino acid sequence at least 70% (e.g., at least75, 80, 85, 90, 95, 98, or 100%) identical to SEQ ID NO:2. If thepolypeptide includes a sequence that is 100% identical to SEQ ID NO:2,the polypeptide can contain up to 30 conservative amino acidsubstitutions. The invention also includes a substantially purepolypeptide encoded by a nucleic acid that hybridizes under stringentconditions to a probe the sequence of which consists of SEQ ID NO:1. Thepolypeptide binds to an androgen receptor and increases the ability ofthe androgen receptor to transactivate an androgen-responsive gene, asshown in the Example below.

The invention further features an isolated nucleic acid encoding apolypeptide of the invention, a vector including a nucleic acid of theinvention, and a cultured host cell containing a nucleic acid of theinvention. An example of a nucleic acid within the invention includes anisolated nucleic acid having a strand that hybridizes under stringentconditions to a single stranded probe, the sequence of which consists ofSEQ ID NO: 1 or the complement of SEQ ID NO:1. Such a nucleic acid canbe at least 15 (e.g., at least 30, 50, 100, 200, 500, or 1000)nucleotides in length.

In addition, the invention features a method of producing a polypeptideby culturing a cultured host cell of the invention in a culture,expressing the polypeptide in the cultured host cell, and isolating thepolypeptide from the culture.

The invention also features a method of screening for a compound thatdecreases androgen receptor-mediated transactivation by contacting apolypeptide of the invention with a protein complex including anandrogen receptor, in the presence of a candidate compound; measuringthe extent of binding between the polypeptide and the protein complex;and determining whether the extent of binding is less than the extent ofbinding between the polypeptide and the protein complex in the absenceof the candidate compound. An extent of binding in the presence of thecompound less than the extent of binding in the absence of the compoundindicates that the candidate compound decreases androgenreceptor-mediated transactivation.

The term “substantially pure” as used herein in reference to a givenpolypeptide means that the polypeptide is substantially free from otherbiological macromolecules. The substantially pure polypeptide is atleast 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight. Puritycan be measured by any appropriate standard method, for example, bycolumn chromatography, polyacrylamide gel electrophoresis, or HPLCanalysis.

A “conservative amino acid substitution” is one in which an amino acidresidue is replaced with another residue having a chemically similarside chain. Families of amino acid residues having similar side chainshave been defined in the art. These families include amino acids withbasic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine).

By hybridization under “stringent conditions” is meant hybridization at65° C., 0.5×SSC, followed by washing at 45° C., 0.1×SSC.

The “percent identity” of two amino acid sequences or of two nucleicacids is determined using the algorithm of Karlin and Altschul (Proc.Natl. Acad. Sci. USA 87:2264–2268, 1990), modified as in Karlin andAltschul (Proc. Natl. Acad. Sci. USA 90:5873–5877, 1993). Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al. (J. Mol. Biol. 215:403–410, 1990). BLAST nucleotidesearches are performed with the NBLAST program, score=100,wordlength=12. BLAST protein searches are performed with the XBLASTprogram, score=50, wordlength=3. Where gaps exist between two sequences,Gapped BLAST is utilized as described in Altschul et al. (Nucleic AcidsRes. 25:3389–3402, 1997). When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) are used. See http://www.ncbi.nlm.nih.gov.

An “isolated nucleic acid” is a nucleic acid the structure of which isnot identical to that of any naturally occurring nucleic acid or to thatof any fragment of a naturally occurring genomic nucleic acid spanningmore than three separate genes. The term therefore covers, for example,(a) a DNA which has the sequence of part of a naturally occurringgenomic DNA molecule but is not flanked by both of the coding sequencesthat flank that part of the molecule in the genome of the organism inwhich it naturally occurs; (b) a nucleic acid incorporated into a vectoror into the genomic DNA of a prokaryote or eukaryote in a manner suchthat the resulting molecule is not identical to any naturally occurringvector or genomic DNA; (c) a separate molecule such as a cDNA, a genomicfragment, a fragment produced by polymerase chain reaction (PCR), or arestriction fragment; and (d) a recombinant nucleotide sequence that ispart of a hybrid gene, i.e., a gene encoding a fusion protein.Specifically excluded from this definition are nucleic acids present inmixtures of different (i) DNA molecules, (ii) transfected cells, or(iii) cell clones, e.g., as these occur in a DNA library such as a cDNAor genomic DNA library.

The polypeptides of the invention can be used to generate antibodies(either monoclonal or polyclonal) that specifically bind to ARCAPprotein. These antibodies in turn are useful for detecting the presenceand distribution of ARCAP in tissues and in cellular compartments. Forexample, such antibodies can be used to diagnose cancerous liver tissueby determining whether ARCAP protein is expressed or overexpressed inthe tissue. Similarly, the nucleic acids of the invention can be used todiagnose liver cancer by determining whether ARCAP mRNA is beingexpressed or overexpressed in a tissue or cell. The nucleic acids can beused as primers in PCR-based detection methods, or as labeled probes innucleic acid blots (e.g., Northern blots).

Other features or advantages of the present invention will be apparentfrom the following detailed description, and also from the claims.

DETAILED DESCRIPTION

The invention relates to new ARCAP proteins and nucleic acids encodingthem that are overexpressed in hepatocellular carcinoma cells relativeto normal liver cells. In addition to differential expression, ARCAP wasfound to bind to and augment the transactivation activity of an androgenreceptor. These observations and others described below suggest thatARCAP activates, via an androgen receptor complex, mitogenic genes thatare androgen-responsive (i.e., genes whose promoters contain androgenresponsive elements), that overexpression of ARCAP leads to cancer byfacilitating androgen receptor-mediated transactivation ofandrogen-responsive mitogenic genes, and that inhibition of ARCAPexpression or activity would reduce expression of theseandrogen-responsive mitogenic genes and revert cancer cells to a morenormal phenotype. Consequently, ARCAP is a new cancer drug target.

Uses

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods: a)screening assays; b) predictive medicine (e.g., diagnostic assays,prognostic assays, monitoring clinical trials, and pharmacogenetics);and c) methods of treatment (e.g., therapeutic and prophylactic).

The isolated nucleic acid molecules of the invention can be used, forexample, to express an ARCAP protein (e.g., via a recombinant expressionvector in a host cell in gene therapy applications), to detect an ARCAPmRNA (e.g., in a biological sample) or a genetic alteration in an ARCAPgene, and to modulate ARCAP activity. The ARCAP proteins can be used totreat disorders characterized by insufficient or excessive production ofan ARCAP substrate or production of ARCAP inhibitors. In addition, theARCAP proteins can be used to screen for naturally occurring ARCAPsubstrates, to screen for drugs or compounds which modulate ARCAPactivity, as well as to treat disorders characterized by insufficient orexcessive production of ARCAP protein or production of ARCAP proteinforms that have decreased, aberrant, or unwanted activity compared toARCAP wild type protein (e.g., in liver cancer). Moreover, theanti-ARCAP antibodies of the invention can be used to detect and isolateARCAP proteins, regulate the bioavailability of ARCAP proteins, andmodulate ARCAP activity.

A method of evaluating a compound for the ability to interact with,e.g., bind, a subject ARCAP polypeptide is provided. The methodincludes: contacting the compound with the subject ARCAP polypeptide;and evaluating ability of the compound to interact with, e.g., to bindor form a complex with, the subject ARCAP polypeptide. This method canbe performed in vitro, e.g., in a cell free system, or in vivo, e.g., ina two-hybrid interaction trap assay. This method can be used to identifynaturally occurring molecules that interact with subject ARCAPpolypeptide. It can also be used to find natural or synthetic inhibitorsof a subject ARCAP polypeptide.

Screening Assays

The invention provides methods (also referred to herein as “screeningassays”) for identifying modulators, i.e., candidate or test compoundsor agents (e.g., proteins, peptides, peptidomimetics, peptoids, smallmolecules, or other drugs) which bind to ARCAP proteins, have astimulatory or inhibitory effect on, for example, ARCAP expression orARCAP activity, or have a stimulatory or inhibitory effect on, forexample, the expression or activity of an ARCAP substrate. Compoundsthus identified can be used to modulate the activity of target geneproducts (e.g., ARCAP genes) in a therapeutic protocol, to elaborate thebiological function of the target gene product, or to identify compoundsthat disrupt normal target gene interactions.

In one embodiment, the invention provides assays for screening candidateor test compounds which are substrates of an ARCAP protein orpolypeptide or a biologically active portion thereof. In anotherembodiment, the invention provides assays for screening candidate ortest compounds which bind to or modulate the activity of an ARCAPprotein or polypeptide or a biologically active portion thereof.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including: biological libraries; peptoid libraries (libraries ofmolecules having the functionalities of peptides, but with a novel,non-peptide backbone which is resistant to enzymatic degradation butwhich nevertheless remains bioactive; see, e.g., Zuckermann, R. N. etal. (1994) J. Med. Chem. 37:2678–85); spatially addressable parallelsolid phase or solution phase libraries; synthetic library methodsrequiring deconvolution; the ‘one-bead one-compound’ library method; andsynthetic library methods using affinity chromatography selection. Thebiological library and peptoid library approaches are limited to peptidelibraries, while the other four approaches are applicable to peptide,non-peptide oligomer, or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412–421), on beads (Lam (1991) Nature354:82–84), chips (Fodor (1993) Nature 364:555–556), bacteria (Ladner,U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc Natl Acad Sci USA 89:1865–1869), or on phage(Scott and Smith (1990) Science 249:386–390; Devlin (1990) Science249:404–406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378–6382;Felici (1991) J. Mol. Biol. 222:301–310; Ladner supra.).

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses an ARCAP protein or biologically active portion thereof iscontacted with a test compound, and the ability of the test compound tomodulate ARCAP activity is determined. Determining the ability of thetest compound to modulate ARCAP activity can be accomplished bymonitoring, for example, cell cycle-regulated cellular localization. Thecell, for example, can be of mammalian origin, e.g., human.

The ability of the test compound to modulate ARCAP binding to acompound, e.g., an androgen receptor complex, or to bind to ARCAP canalso be evaluated. This can be accomplished, for example, by couplingthe compound, e.g., the substrate, with a radioisotope or enzymaticlabel such that binding of the compound, e.g., the substrate, to ARCAPcan be determined by detecting the labeled compound, e.g., substrate, ina complex. Alternatively, ARCAP could be coupled with a radioisotope orenzymatic label to monitor the ability of a test compound to modulateARCAP binding to an ARCAP substrate in a complex. For example, compounds(e.g., ARCAP substrates) can be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H,either directly or indirectly, and the radioisotope detected by directcounting of radioemmission or by scintillation counting. Alternatively,compounds can be enzymatically labeled with, for example, horseradishperoxidase, alkaline phosphatase, or luciferase, and the enzymatic labeldetected by determination of conversion of an appropriate substrate toproduct.

The ability of a compound (e.g., an ARCAP substrate) to interact withARCAP with or without the labeling of any of the interactants can beevaluated. For example, a microphysiometer can be used to detect theinteraction of a compound with ARCAP without the labeling of either thecompound or the ARCAP. McConnell, H. M. et al. (1992) Science257:1906–1912. As used herein, a “microphysiometer” (e.g., Cytosensor)is an analytical instrument that measures the rate at which a cellacidifies its environment using a light-addressable potentiometricsensor (LAPS). Changes in this acidification rate can be used as anindicator of the interaction between a compound and ARCAP.

In yet another embodiment, a cell-free assay is provided in which anARCAP protein or biologically active portion thereof is contacted with atest compound and the ability of the test compound to bind to the ARCAPprotein or biologically active portion thereof is evaluated. Preferredbiologically active portions of the ARCAP proteins to be used in assaysof the present invention include fragments which participate ininteractions with non-ARCAP molecules, e.g., fragments with high surfaceprobability scores.

Soluble and/or membrane-bound forms of isolated proteins (e.g., ARCAPproteins or biologically active portions thereof) can be used in thecell-free assays of the invention. When membrane-bound forms of theprotein are used, it may be desirable to utilize a solubilizing agent.Examples of such solubilizing agents include non-ionic detergents suchas n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100,Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)_(n,)3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate.

Cell-free assays involve preparing a reaction mixture of the target geneprotein and the test compound under conditions and for a time sufficientto allow the two components to interact and bind, thus forming a complexthat can be removed and/or detected.

The interaction between two molecules can also be detected, e.g., usingfluorescence energy transfer (FET) (see, for example, Lakowicz et al.,U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No.4,868,103). A fluorophore label on the first, ‘donor’ molecule isselected such that its emitted fluorescent energy will be absorbed by afluorescent label on a second, ‘acceptor’ molecule, which in turn isable to fluoresce due to the absorbed energy. Alternately, the ‘donor’protein molecule may simply utilize the natural fluorescent energy oftryptophan residues. Labels are chosen that emit different wavelengthsof light, such that the ‘acceptor’ molecule label may be differentiatedfrom that of the ‘donor.’ Since the efficiency of energy transferbetween the labels is related to the distance separating the molecules,the spatial relationship between the molecules can be assessed. In asituation in which binding occurs between the molecules, the fluorescentemission of the ‘acceptor’ molecule label in the assay should bemaximal. An FET binding event can be conveniently measured throughstandard fluorometric detection means well known in the art (e.g., usinga fluorimeter).

In another embodiment, determining the ability of the ARCAP protein tobind to a target molecule can be accomplished using real-timeBiomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. andUrbaniczky, C. (1991) Anal. Chem. 63:2338–2345 and Szabo et al. (1995)Curr. Opin. Struct. Biol. 5:699–705). “Surface plasmon resonance” or“BIA” detects biospecific interactions in real time, without labelingany of the interactants (e.g., BIAcore). Changes in the mass at thebinding surface (indicative of a binding event) result in alterations ofthe refractive index of light near the surface (the optical phenomenonof surface plasmon resonance (SPR)), resulting in a detectable signalwhich can be used as an indication of real-time reactions betweenbiological molecules.

In one embodiment, the target gene product or the test substance isanchored onto a solid phase. The target gene product/test compoundcomplexes anchored on the solid phase can be detected at the end of thereaction. Preferably, the target gene product can be anchored onto asolid surface, and the test compound, (which is not anchored), can belabeled, either directly or indirectly, with detectable labels discussedherein.

It may be desirable to immobilize either ARCAP, an anti-ARCAP antibodyor its target molecule to facilitate separation of complexed fromuncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay. Binding of a test compound to anARCAP protein, or interaction of an ARCAP protein with a target moleculein the presence and absence of a candidate compound, can be accomplishedin any vessel suitable for containing the reactants. Examples of suchvessels include microtiter plates, test tubes, and micro-centrifugetubes. In one embodiment, a fusion protein can be provided which adds adomain that allows one or both of the proteins to be bound to a matrix.For example, glutathione-S-transferase/ARCAP fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or ARCAP protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotiter plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads. Complexes are determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level of ARCAPbinding or activity determined using standard techniques.

Other techniques for immobilizing either an ARCAP protein or a targetmolecule on matrices include using conjugation of biotin andstreptavidin. Biotinylated ARCAP protein or target molecules can beprepared from biotin-NHS(N-hydroxy-succinimide) using techniques knownin the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL),and immobilized in the wells of streptavidin-coated 96 well plates(Pierce Chemical).

In order to conduct the assay, the non-immobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynon-immobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously non-immobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the immobilized component (theantibody, in turn, can be directly labeled or indirectly labeled with,e.g., a labeled anti-Ig antibody).

In one embodiment, this assay is performed utilizing antibodies reactivewith ARCAP protein or target molecules but which do not interfere withbinding of the ARCAP protein to its target molecule. Such antibodies canbe derivatized to the wells of the plate, and unbound target or ARCAPprotein trapped in the wells by antibody conjugation. Methods fordetecting such complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with the ARCAP protein or target molecule, as wellas enzyme-linked assays which rely on detecting an enzymatic activityassociated with the ARCAP protein or target molecule.

Alternatively, cell free assays can be conducted in a liquid phase. Insuch an assay, the reaction products are separated from unreactedcomponents by any of a number of standard techniques including but notlimited to differential centrifugation (see, for example, Rivas, G., andMinton, A. P., (1993) Trends Biochem Sci 18:284–7); chromatography (gelfiltration chromatography, ion-exchange chromatography); electrophoresis(see, e.g., Ausubel, F. et al., eds. Current Protocols in MolecularBiology 1999, J. Wiley: New York.); and immunoprecipitation (see, forexample, Ausubel, F. et al., eds. (1999) Current Protocols in MolecularBiology, J. Wiley: New York). Such resins and chromatographic techniquesare known to one skilled in the art (see, e.g., Heegaard, N. H., (1998)J Mol Recognit 11:141–8; Hage, D. S., and Tweed, S. A. (1997) JChromatogr B Biomed Sci Appl. 699:499–525). Further, fluorescence energytransfer may also be conveniently utilized, as described herein, todetect binding without further purification of the complex fromsolution.

In a preferred embodiment, the assay includes contacting the ARCAPprotein or biologically active portion thereof with a known compound(e.g., an androgen receptor) which binds ARCAP to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with an ARCAP protein, wheredetermining the ability of the test compound to interact with an ARCAPprotein includes determining the ability of the test compound topreferentially bind to ARCAP or biologically active portion thereof, orto modulate the activity of a target molecule, as compared to the knowncompound.

The target gene products of the invention can, in vivo, interact withone or more cellular or extracellular macromolecules, such as proteins.For the purposes of this discussion, such cellular and extracellularmacromolecules are referred to herein as “binding partners.” Compoundsthat disrupt such interactions can be useful in regulating the activityof the target gene product. Such compounds can include, but are notlimited to molecules such as antibodies, peptides, and small molecules.The preferred target genes/products for use in this embodiment are theARCAP genes herein identified. In an alternative embodiment, theinvention provides methods for determining the ability of the testcompound to modulate the activity of an ARCAP protein through modulationof the activity of a downstream effector of an ARCAP target molecule.For example, the activity of the effector molecule on an appropriatetarget can be determined, or the binding of the effector to anappropriate target can be determined, as previously described.

To identify compounds that interfere with the interaction between thetarget gene product and its cellular or extracellular bindingpartner(s), a reaction mixture containing the target gene product andthe binding partner is prepared, under conditions and for a timesufficient, to allow the two products to form a complex. In order totest an inhibitory agent, the reaction mixture is provided in thepresence and absence of the test compound. The test compound can beinitially included in the reaction mixture, or can be added at a timesubsequent to the addition of the target gene and its cellular orextracellular binding partner. Control reaction mixtures are incubatedwithout the test compound or with a placebo. The formation of anycomplexes between the target gene product and the cellular orextracellular binding partner is then detected. The formation of acomplex in the control reaction, but not in the reaction mixturecontaining the test compound, indicates that the compound interfereswith the interaction of the target gene product and the interactivebinding partner. Additionally, complex formation within reactionmixtures containing the test compound and normal target gene product canalso be compared to complex formation within reaction mixturescontaining the test compound and mutant target gene product. Thiscomparison can be important in those cases where it is desirable toidentify compounds that disrupt interactions of mutant but not normaltarget gene products.

These assays can be conducted in a heterogeneous or homogeneous format.Heterogeneous assays involve anchoring either the target gene product orthe binding partner onto a solid phase, and detecting complexes anchoredon the solid phase at the end of the reaction. In homogeneous assays,the entire reaction is carried out in a liquid phase. In eitherapproach, the order of addition of reactants can be varied to obtaindifferent information about the compounds being tested. For example,test compounds that interfere with the interaction between the targetgene products and the binding partners, e.g., by competition, can beidentified by conducting the reaction in the presence of the testsubstance. Alternatively, test compounds that disrupt preformedcomplexes, e.g., compounds with higher binding constants that displaceone of the components from the complex, can be tested by adding the testcompound to the reaction mixture after complexes have been formed. Thevarious formats are briefly described below.

In a heterogeneous assay system, either the target gene product or theinteractive cellular or extracellular binding partner is anchored onto asolid surface (e.g., a microtiter plate), while the non-anchored speciesis labeled either directly or indirectly. The anchored species can beimmobilized by non-covalent or covalent attachments. Alternatively, animmobilized antibody specific for the species to be anchored can be usedto anchor the species to the solid surface.

In order to conduct the assay, the partner of the immobilized species isexposed to the coated surface with or without the test compound. Afterthe reaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed will remain immobilized on the solidsurface. Where the non-immobilized species is pre-labeled, the detectionof label immobilized on the surface indicates that complexes wereformed. Where the non-immobilized species is not pre-labeled, anindirect label can be used to detect complexes anchored on the surface,e.g., using a labeled antibody specific for the initiallynon-immobilized species. The antibody, in turn, can be directly labeledor indirectly labeled with, e.g., a labeled anti-Ig antibody. Dependingupon the order of addition of reaction components, test compounds thatinhibit complex formation or that disrupt preformed complexes can bedetected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected, e.g., usingan immobilized antibody specific for one of the binding components toanchor any complexes formed in solution and a labeled antibody specificfor the other partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds that inhibit complex formation or that disrupt preformedcomplexes can be identified.

In an alternate embodiment, a homogeneous assay can be used. Forexample, a preformed complex of the target gene product and theinteractive cellular or extracellular binding partner product isprepared so that either the target gene products or their bindingpartners are labeled, but the signal generated by the label is quencheddue to complex formation (see, e.g., U.S. Pat. No. 4,109,496 thatutilizes this approach for immunoassays). The addition of a testsubstance that competes with and displaces one of the species from thepreformed complex will result in the generation of a signal abovebackground. In this way, test substances that disrupt target geneproduct-binding partner interaction can be identified.

In yet another aspect, the ARCAP proteins can be used as “bait proteins”in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No.5,283,317; Zervos et al. (1993) Cell 72:223–232; Madura et al. (1993) J.Biol. Chem. 268:12046–12054; Bartel et al. (1993) Biotechniques14:920–924; Iwabuchi et al. (1993) Oncogene 8:1693–1696; and BrentWO94/10300) to identify other proteins, which bind to or interact withARCAP (“ARCAP-binding proteins” or “ARCAP-bp”) and are involved in ARCAPactivity. Such ARCAP-bps can be activators or inhibitors of signals bythe ARCAP proteins or ARCAP targets as, for example, downstream elementsof an ARCAP-mediated signaling pathway.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for an ARCAP proteinis fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. (Alternatively, theARCAP protein can be fused to the activator domain.) If the “bait” andthe “prey” proteins are able to interact, in vivo, forming anARCAP-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., lacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinthat interacts with the ARCAP protein.

In another embodiment, modulators of ARCAP expression are identified.For example, a cell or cell free mixture is contacted with a candidatecompound and the expression of ARCAP mRNA or protein evaluated relativeto the level of expression of ARCAP mRNA or protein in the absence ofthe candidate compound. When expression of ARCAP mRNA or protein isgreater in the presence of the candidate compound than in its absence,the candidate compound is identified as a stimulator of ARCAP mRNA orprotein expression. Alternatively, when expression of ARCAP mRNA orprotein is less (statistically significantly less) in the presence ofthe candidate compound than in its absence, the candidate compound isidentified as an inhibitor of ARCAP mRNA or protein expression. Thelevel of ARCAP mRNA or protein expression can be determined by methodsdescribed herein for detecting ARCAP mRNA or protein.

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulating agent canbe identified using a cell-based or a cell free assay, and the abilityof the agent to modulate the activity of an ARCAP protein can beconfirmed in vivo, e.g., in an animal such as an animal model forhepatocellular carcinoma.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein(e.g., an ARCAP modulating agent, an antisense ARCAP nucleic acidmolecule, an ARCAP-specific antibody, or an ARCAP-binding partner) in anappropriate animal model to determine the efficacy, toxicity, sideeffects, or mechanism of action, of treatment with such an agent.Furthermore, novel agents identified by the above-described screeningassays can be used for treating cancers, e.g., liver cancer.

Use of ARCAP Molecules as Surrogate Markers

The ARCAP molecules of the invention are also useful as markers ofdisorders or disease states, as markers for precursors of diseasestates, as markers for predisposition of disease states, as markers ofdrug activity, or as markers of the pharmacogenomic profile of asubject. Using the methods described herein, the presence, absenceand/or quantity of the ARCAP molecules of the invention may be detected,and may be correlated with one or more biological states in vivo. Forexample, the ARCAP molecules of the invention may serve as surrogatemarkers for one or more disorders or disease states or for conditionsleading up to disease states. As used herein, a “surrogate marker” is anobjective biochemical marker which correlates with the absence orpresence of a disease or disorder, or with the progression of a diseaseor disorder (e.g., with the presence or absence of a liver tumor). Thepresence or quantity of such markers is independent of the disease.Therefore, these markers may serve to indicate whether a particularcourse of treatment is effective in lessening a disease state ordisorder. Surrogate markers are of particular use when the presence orextent of a disease state or disorder is difficult to assess throughstandard methodologies (e.g., early stage tumors), or when an assessmentof disease progression is desired before a potentially dangerousclinical endpoint is reached (e.g., an assessment of cardiovasculardisease may be made using cholesterol levels as a surrogate marker, andan analysis of HIV infection may be made using HIV RNA levels as asurrogate marker, well in advance of the undesirable clinical outcomesof myocardial infarction or fully-developed AIDS). Examples of the useof surrogate markers in the art include those described in Koomen et al.(2000) J. Mass. Spectrom. 35: 258–264; and James (1994) AIDS TreatmentNews Archive 209.

The ARCAP molecules of the invention are also useful as pharmacodynamicmarkers. As used herein, a “pharmacodynamic marker” is an objectivebiochemical marker which correlates specifically with drug effects. Thepresence or quantity of a pharmacodynamic marker is not related to thedisease state or disorder for which the drug is being administered;therefore, the presence or quantity of the marker is indicative of thepresence or activity of the drug in a subject. For example, apharmacodynamic marker may be indicative of the concentration of thedrug in a biological tissue, in that the marker is either expressed ortranscribed or not expressed or transcribed in that tissue inrelationship to the level of the drug. In this fashion, the distributionor uptake of the drug may be monitored by the pharmacodynamic marker.Similarly, the presence or quantity of the pharmacodynamic marker may berelated to the presence or quantity of the metabolic product of a drug,such that the presence or quantity of the marker is indicative of therelative breakdown rate of the drug in vivo. Pharmacodynamic markers areof particular use in increasing the sensitivity of detection of drugeffects, particularly when the drug is administered in low doses. Sinceeven a small amount of a drug may be sufficient to activate multiplerounds of marker (e.g., an ARCAP marker) transcription or expression,the amplified marker may be in a quantity which is more readilydetectable than the drug itself. Also, the marker may be more easilydetected due to the nature of the marker itself; for example, using themethods described herein, anti-ARCAP antibodies may be employed in animmune-based detection system for an ARCAP protein marker, orARCAP-specific radiolabeled probes may be used to detect an ARCAP mRNAmarker. Furthermore, the use of a pharmacodynamic marker may offermechanism-based prediction of risk due to drug treatment beyond therange of possible direct observations. Examples of the use ofpharmacodynamic markers in the art are described in Matsuda et al. U.S.Pat. No. 6,033,862; Hattis et al. (1991) Env. Health Perspect. 90:229–238; Schentag (1999) Am. J. Health-Syst. Pharm. 56 Suppl. 3:S21–S24; and Nicolau (1999) Am, J. Health-Syst. Pharm. 56 Suppl. 3:S16–S20.

The ARCAP molecules of the invention are also useful as pharmacogenomicmarkers. As used herein, a “pharmacogenomic marker” is an objectivebiochemical marker which correlates with a specific clinical drugresponse or susceptibility in a subject (see, e.g., McLeod et al. (1999)Eur. J. Cancer 35:1650–1652). The presence or quantity of thepharmacogenomic marker is related to the predicted response of thesubject to a specific drug or class of drugs prior to administration ofthe drug. By assessing the presence or quantity of one or morepharmacogenomic markers in a subject, a drug therapy which is mostappropriate for the subject, or which is predicted to have a greaterdegree of success, may be selected. For example, based on the presenceor quantity of RNA, or protein (e.g., ARCAP protein or RNA) for specifictumor markers in a subject, a drug or course of treatment may beselected which is optimized for the treatment of the specific tumorlikely to be present in the subject. Similarly, the presence or absenceof a specific sequence mutation in ARCAP DNA may correlate with ARCAPdrug response. The use of pharmacogenomic markers therefore permits theapplication of the most appropriate treatment for each subject withouthaving to administer the therapy.

Pharmacogenomics

The ARCAP molecules of the present invention, as well as agents, ormodulators which have a stimulatory or inhibitory effect on ARCAPactivity (e.g., ARCAP gene expression) as identified by a screeningassay described herein can be administered to individuals to treat(prophylactically or therapeutically) ARCAP associated disorders (e.g.,liver cancer) associated with aberrant or unwanted ARCAP activity. Inconjunction with such treatment, pharmacogenomics (i.e., the study ofthe relationship between an individual's genotype and that individual'sresponse to a foreign compound or drug) may be considered. Differencesin metabolism of therapeutics can lead to severe toxicity or therapeuticfailure by altering the relation between dose and blood concentration ofthe pharmacologically active drug. Thus, a physician or clinician mayconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer an ARCAP molecule or ARCAPmodulator as well as tailoring the dosage and/or therapeutic regimen oftreatment with an ARCAP molecule or ARCAP modulator.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, for example, Eichelbaum, M. et al.(1996) Clin. Exp. Pharmacol. Physiol. 23:983–985 and Linder, M. W. etal. (1997) Clin. Chem. 43:254–266. In general, two types ofpharmacogenetic conditions can be differentiated. Genetic conditionstransmitted as a single factor altering the way drugs act on the body(altered drug action) or genetic conditions transmitted as singlefactors altering the way the body acts on drugs (altered drugmetabolism). These pharmacogenetic conditions can occur either as raregenetic defects or as naturally-occurring polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

One pharmacogenomics approach to identifying genes that predict drugresponse, known as “a genome-wide association,” relies primarily on ahigh-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000–100,000 polymorphic or variable sites on the humangenome, each of which has two variants). Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high resolution map can begenerated from a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP may occur once per every 1000 bases of DNA. ASNP may be involved in a disease process, however, the vast majority maynot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that may becommon among such genetically similar individuals.

Alternatively, a method termed the “candidate gene approach”, can beutilized to identify genes that predict drug response. According to thismethod, if a gene that encodes a drug's target is known (e.g., an ARCAPprotein of the present invention), all common variants of that gene canbe fairly easily identified in the population, and it can be determinedif having one version of the gene versus another is associated with aparticular drug response.

Alternatively, a method termed the “gene expression profiling”, can beutilized to identify genes that predict drug response. For example, thegene expression of an animal dosed with a drug (e.g., an ARCAP moleculeor ARCAP modulator of the present invention) can give an indicationwhether gene pathways related to toxicity have been turned on.

Information generated from more than one of the above pharmacogenomicsapproaches can be used to determine appropriate dosage and treatmentregimens for prophylactic or therapeutic treatment of an individual.This knowledge, when applied to dosing or drug selection, can avoidadverse reactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with an ARCAP moleculeor ARCAP modulator, such as a modulator identified by one of theexemplary screening assays described herein.

The present invention further provides methods for identifying newagents, or combinations, that are based on identifying agents thatmodulate the activity of one or more of the gene products encoded by oneor more of the ARCAP genes of the present invention, where theseproducts may be associated with resistance of the cells to a therapeuticagent. Specifically, the activity of the proteins encoded by the ARCAPgenes of the present invention can be used as a basis for identifyingagents for overcoming agent resistance. By blocking the activity of oneor more of the resistance proteins, target cells, e.g., human cells,will become sensitive to treatment with an agent that the unmodifiedtarget cells were resistant to.

Monitoring the influence of agents (e.g., drugs) on the expression oractivity of an ARCAP protein can be applied in clinical trials. Forexample, the effectiveness of an agent determined by a screening assayas described herein to increase ARCAP gene expression, protein levels,an ARCAP activity can be monitored in clinical trials of subjectsexhibiting decreased ARCAP gene expression, protein levels, or ARCAPactivity. Alternatively, the effectiveness of an agent determined by ascreening assay to decrease ARCAP gene expression, protein levels, or anARCAP activity can be monitored in clinical trials of subjectsexhibiting increased ARCAP gene expression, protein levels, or ARCAPactivity. In such clinical trials, the expression or activity of anARCAP gene, and preferably other genes that have been implicated in, forexample, an ARCAP-associated disorder can be used as a “read out” ormarker of the phenotype of a particular cell.

Without further elaboration, it is believed that one skilled in the artcan, based on the above disclosure and the example below, utilize thepresent invention to its fullest extent. The following examples are tobe construed as merely illustrative of how one skilled in the art canisolate and use the polypeptides and nucleic acids of the invention, andare not limitative of the remainder of the disclosure in any way. Anypublications cited in this disclosure are hereby incorporated byreference.

EXAMPLE

Materials and Methods

Patient samples. Hepatocellular carcinoma patients from the Departmentof Surgery, Veterans General Hospital, Taipei, Taiwan, were recruitedinto this study. Diagnosis of hepatoma was made by sonography,angiography, computer tomography, and/or magnetic resonance imaging.Clinical information for each patient was recorded, including thepatients' age, sex, serum alpha-fetoprotein level, liver function, tumorsize, tumor location, pathological staging, disease-free interval, timeof recurrence, and location of tumor recurrence. Informed consent wasobtained from each patient. For each liver cancer patient, tissue washarvested from the tumor, as well as from the normal liver tissue thatwas adjacent to the tumor.

RNA extraction and reverse transcription for complement DNA. Tissuespecimens were frozen immediately after surgical resection and stored at−80° C. RNA was extracted using acid guanidinium thiocyanate andphenol/chloroform extraction as described in Chomczynski et al. (1987)Anal. Biochem. 162:156–159. Approximately 0.5 g of frozen tissue washomogenized with 4 ml RNAzol B (Biotecx Laboratories, Houston, Tex.)using a polytron. DNA was sheared using a Douncer with a type-B pestle.After adding 0.4 ml of chloroform, vigorous vortexing, and standing onice for 5 minutes, the mixture was separated by centrifugation at 12,000g at 4° C. for 15 minutes. The upper aqueous phase containing total RNAwas precipitated with an equal volume of isopropanol.

Complement DNA was synthesized from the 1 μg of total RNA. Reversetranscription was performed in a volume of 30 μl, containing RNA and 1×first strand buffer (10 mM DTT, 500 μM dNTPs, 50 ng/ml oligo-dT, and 100units MMLV reverse transcriptase) at 37° C. for 1 hour (LifeTechnologies). The samples were then denatured at 95° C. for 5 minutes.

PCR amplification. cDNA (1 μl) was PCR amplified in a volume of 25 μlcontaining 0.8 μM of primers, 50 μM of each dNTP (Takara), 1×PCR buffer,and 1.25 units Taq Polymerase (Pharmacia). As a control for cDNAquality, a test PCR reaction was carried out using the human transferringene primers Tref8 (GGAACATTTTGGCAAAGACA [SEQ ID NO:4]; derived fromnucleotides 971–990 of the transferring cDNA sequence) and Tref9(ATTCATGATCTT(C/T)GCGATGC [SEQ ID NO:5]; derived from nucleotides1307–1288 of the transferrin cDNA sequence). These primer sequences werechosen form the eighth and ninth exons of the human transferrin gene,respectively. PCR was performed using an initial denaturation at 94° C.for 10 minutes; followed by 30 cycles of 94° C. for 1 minutes, 55° C.for 1 minute, and 72° C. for 1 minute; then a final extension at 72° C.for 10 minutes. A successful PCR yielded a 336 bp product, indicatingthat the cDNA template is at least 1.4 kb from the poly(A) end (nt2362).

The steroid receptor superfamily clones were generated by aminoacid-based homology PCR using degenerate primers encoding highlyconserved sequence motifs in the zinc finger of DNA binding domain ofsteroid receptors. The primers encoded the amino acid sequences DYSTGYHY(SEQ ID NO:6), CKXFFKR (SEQ ID NO:7), and CPACRFXKC (SEQ ID NO:8), allof which are described in Maksymowych et al (1992) Receptor 2:225–240.The forward and reverse primer sequences wereRCAYTTIIIIARICKRCAIKMNKGRCA (SEQ ID NO:11), and GAYRARKClWCIGGIWRICAYT(SEQ ID NO:12). PCR was performed under low stringencyannealing/extension conditions (42° C./65° C.) for 5 cycles, beforeamplifying the templates at high-stringency conditions (55° C./72° C.).The PCR products were subcloned into the TA vector (Invitrogen), and theresulting plasmid used to transform DH5α cells. Clones were randomlypicked and checked for an insert size of about 170 bp, a lengthcorresponding to a zinc finger. A high frequency of clones containedappropriately sized inserts (85–90%).

DNA Sequence Analysis of novel ARCAP. Positive clones were picked andsequenced using Applied Biosystems model 377 DNA sequencers. The clonedsequences were analyzed using the BLASTN program (Zehetner et al. (1994)Nature 367:489–491). Partial cDNA clones bearing sequence similarity tomembers of the steroid receptor superfamily were selected for furtherstudy. Of these clones, one clone named ARCAP, was characterized andfully cloned as described herein.

Isolation of full length clone. To obtain the complete open readingframe of ARCAP, a commercial cDNA library of G2 hepatoma cell line(Clontech) was probed with the ARCAP partial clone. In addition,hepatoma cDNA libraries were constructed from RNA isolated from ahepatoma tumor and normal human liver tissue, both donated by a malepatient that died from trauma. Approximately 5 μg of mRNA was used forreverse transcription. The cDNA library was prepared using the lambdaZAP II system (Stratagene). The library was amplified from 1.1×10⁶ PFUof primary recombinant clones. The average insert size was 1.2 kb. Morethan 95% of the clones are recombinants.

To isolate novel clones from the liver cDNA libraries, radio-labeledprobes were prepared using a PCR reaction supplemented with a³²P-labeled dCTP. Specifically, a labeling reaction of 100 μl volumecontained 1× buffer (1.5 μM MgCl₂, 0.5 μl Taq polymerase (25 units/ml),200 μM each of dGTP, dTTP, and dATP, and 25–50 μM dCTP) and 5 μl of³²P-α-dCTP. The PCR conditions for producing labeled probes wasessentially the same as described above for amplifying steroid receptorclones based on conserved domains.

The cDNA library was screened at a moderate density (16,000 PFU per 150mm plate). Twenty plates were initially screened. Pre-hybridization wascarried out in 5×SSC, 2× Denhardt's, 100 mg/ml single-stranded salmonsperm DNA, and 0.1% SDS at 55° C. Hybridization was performed in thesame solution but supplemented with 1×10⁷ cpm/ml solution of denaturedPCR-labeled probes. After incubation at 55° C. for 20 hours, lowstringency washing was performed for 60 minutes at room temperatureusing 2×SSC, 0.1% SDS. The blots were visualized by autoradiography (24hours exposure) using Kodak X-AR film and one intensifying screen(Lighting Plus).

Cloning by 5′-RACE and 3′-RACE. RACE is a procedure for amplification ofa cDNA template between a known internal site and unknown sequences ateither 5′ or the 3′ ends (Fronhman et al., (1988) Proc. Natl. Acad. Sci.USA 85:8998–9001). The basic protocol performed in this study wasdescribed in the literature accompanying Clontech's RACE kits.

GSP1 (TCTGGTGGTTGCACTGAGCT; SEQ ID NO:13) and GSP2 (ACAATGTCAGCTCAGGCTC;SEQ ID NO:14) primers were designed in-house and based on the sequenceof ARCAP. Human hepatoma cell line G2 mRNA was used as template forsynthesis of first strand cDNA. This synthesis was performed usingprimers 3′-CDS (AAGCAGTGGTAACAACGCAGAGTACT₃₀ NN; SEQ ID NO:15) for 3′RACE or Smart-oligo (T₂₅NN; SEQ ID NO:16) plus 5′-CDS(AAGCAGTGGTAACAACGCAGAG TACGCGGG; SEQ ID NO:17) for 5′ RACE.

After synthesis of first strand cDNA, 5′ RACE was performed by PCR usingSmart and GSP1 primers. For 3′ RACE, PCR was performed using GSP2 and3′CDS primers. Each fragment after PCR were cloning into pGEM-easyvector (Promega). After screening, the correct clones were picked, andplasmid DNAs were purified. The full length ARCAP cDNA sequence was thusobtained.

Production of ARCAP antibody. The ARCAP clone was digested with EcoR1and inserted into pGEX2T to produce a ARCAP-GST expression plasmid(Pharmacia). This plasmid was used to transform BL21 bacteria, whichwere then induced using IPTG to express the fusion protein. The fusionprotein was purified from bacterial lysate using Glutathione Sepharose4B affinity chromatography. The ARCAP protein was detected by Westernblotting using an anti-GST antibody. Twenty micrograms of fusion proteinwas injected into Balb/c mice to raise ARCAP antiserum.

ARCAP expression analysis. To determine the mRNA expression pattern forARCAP, RNA samples prepared from tissues and cell lines were analyzed byNorthern blot. Twenty micrograms each of total RNA from liver, fetalbrain, four cell lines derived from liver (HepG2, Hep3B, VGH/22T,VGH/59T), blood cell lines (K562, U932, Ramos, Jurkat), and HeLa cellswere separated on a formaldehyde gel and transferred to a filtermembrane. Filter hybridization was carried out with the labeled probesat 42° C. in 5×SSC, 5× Denhardt's, 5 mg/ml denatured salmon sperm DNA,50% formamide, and 0.1% SDS. Washing was first performed at roomtemperature in 2×SSC/0.1% SDS for 30 minutes, followed by washing at 42°C. in 2×SSC/0.1% SDS for 30 minutes, and then a final washing at 55° C.in 0.2×SSC/0.1% SDS for 30 minutes. Autoradiography film was exposedusing one intensifying screen for 72 hours.

For in situ hybridization, liver cancer tissue sections were obtainedfrom the patient samples described above. A riboprobe of ARCAP wasprepared using the partial ARCAP clone and a biotin labeling kit (NEN).Hybridization and washes were carried out according to establishedprotocols. For ARCAP protein expression, an antibody was raised using aGST-ARCAP fusion expression vector as described herein. This antibodywas then used to identify the presence of ARCAP protein in the varioustissue sections.

ARCAP transactivation of androgen-responsive genes. COS-1 cells werecultured for at least 48 hours before transfection in Dulbecco'smodified Eagle's medium (Life Technologies, Inc.) supplemented with 10%fetal calf serum that had been stripped of steroids by treatment withdextran-coated charcoal. Cells were grown to 60–80% confluence, washed,removed, and seeded at a density of 5×10⁴ cells/well in fresh medium in35 mm wells (Corning) of a microtitre tissue culture plate. After 20hours, cells were washed once with serum-free medium and transfectedusing Fugene 6 (Roche) according to the manufacturer's directions. Tenhours thereafter, cells were washed twice with the appropriate mediumand incubated in 2 ml of medium containing steroid or vehicle. After 48hours, cells were recovered and assayed for luciferase activityaccording to the manufacturer's instructions (Promega).

Luciferase activity was corrected for the corresponding β-galactosidaseactivity to give relative activity. β-Galactosidase assays wereperformed in a 96-well plate (Corning) as follows: 10 μl of sampleextract were incubated with 80 μl of buffer Z and 10 μl ofo-nitrophenyl-β-D-galactopyranoside (4 mg/ml) at 30° C. for 2 hours. Thereaction was terminated by the addition of 50 μl of 1 M Na₂CO₃. A₄₂₀values were obtained using a MR5000 plate reader (Dynatech), andactivity was calculated as described herein. Transfections wereperformed in triplicate and repeated at least three times.

Hepatoma A2 cells were subcultured in DMEM medium (Life Technologies,Inc.) supplemented with 10% fetal calf serum. Cells were seeded at 1×10⁵cells/well in 35 mm wells at least 24 hours before transfection. Forstudies examining the interaction between AR and ARCAP-1, cells werecultured in DMEM supplemented with 10% dextran-coated charcoal-fetalcalf serum. Cells were transfected using Fugene 6 for a period of 8hours, washed, and cultured in medium containing vehicle or steroid.Cells were harvested at 48 hours after transfection and analyzed asdescribed above.

A 450 base fragment containing an androgen-responsive element(TGGGTACATTTTGTTC; SEQ ID NO:9) from a portion of the humanalpha-fetoprotein promoter 4385 bp from the ATG start coding site(Genbank ID: G178242) was found to have transactivation response abilitywhen cloned into a heterologous gene. This fragment was amplified by PCRfrom human genomic DNA (Clontech). A element androgen responsive elementcontaining the sequence TGGGTAGGTTTTGCTC (SEQ ID NO:10) was made by sitedirect mutagenesis. The mutated nucleotides are indicated byunderlining. This product was cloned into the pGEM-easy vector(Promega), and the sequence of the element was confirmed by sequencing.The inserted DNA were subcloned into the appropriate sites in theluciferase reporter plasmid pGL3basic (Promega).

AR and ARCAP were fused in frame to the amino terminus of enhanced GFP(EGFP) in the vector pEGFP—N1 (Clontech) using an EcoRI site. Thisplasmid was used to transform into E. coli (DH5). Positive clones wereidentified using mini-plasmid preparation and restriction enzymeanalysis. Selected constructs were confirmed by sequencing. COS7 and A2cells were cultured in DMEM supplemented with 10% fetal calf serum,penicillin/streptomycin (100 μg/ml), and L-glutamine (2 mM).Subconfluent monolayers (10⁶ cells in 100 mm dishes) were transfectedwith 10 μg of AR and ARCAP cDNA by calcium phosphate. Thirty hourslater, fresh media were replaced with medium supplemented with DHT (10nM). The cells were observed using a fluorescence microscope (Nikon).

Immunoprecipitations. Expression plasmids encoding androgen receptor,ARA70, and ARCAP-HA or ARCAP-Xpress epitope fusion protein were producedin pCDNA III (Invitrogen) using EcoRI. After transfection into COS7cells, protein was labeled using the in vitro-coupled transcription andtranslation kit, T7-TNT (Promega), for incorporating ³⁵S-methione. At 48hours post-transfection, the cultures were supplemented to 10 nM DHT.Cells were lysed, and the lysate was incubated with 1 ml ofimmunoprecipitation buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.2 mMNa₃VO₄, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 mMdithiothreitol, 25 μg/ml leupeptin, 25 μg/ml aprotinin, and 25 μg/mlpepstatin). The lysate was then mixed and incubated with AR, HA, orXpress antibody (Invitrogen) on ice for 60 minutes, at which time 10 μlof protein A-sepharose beads (Pharmacia) were added. Theimmunoprecipitations were incubated for 16 hours at 4° C. withcontinuous mixing. Immunoprecipitated complexes were collected bycentrifugation at 2000 rpm at 4° C. for 10 minutes. The pelleted beadswere washed three times and mixed with SDS sample buffer. Samples wereresolved on 8% polyacrylamide gels at 200 V for 45 minutes. The gel wasfixed for 30 minutes in 10% propanol and 10% acetic acid, soaked inAmplify (Amersham Pharmacia Biotech) for 30 minutes, dried under vacuum,and exposed to X-ray film for 4 to 24 hours at 70° C.

Yeast two hybrid analysis. For independent cloning of ARCAP, androgenreceptor was used as a bait to screen the matchmaker yeast two hybridlibrary (Clontech). Briefly, the full length insert of androgen receptorsequence was cloned into pS2-1 (Clontech), and the resulting constructused to screen a yeast-two hybrid testis library constructed in pACT2(Clontech), using the yeast host Y187, according to the manufacturer'sprotocols. Two hundred thousand transformed cells were plated onto medialacking tryptophan, leucine, and adenine, but supplement with 1 μMdihydrotestosterone (DHT). Adenine-positive, LacZ-positive,histidine-positive colonies were isolated, and plasmid DNA obtainedtherefrom. The plasmids were transformed into E. coli.

pACT2 plasmids containing cDNA were identified by colony PCR usingprimers specific for the LEU2 gene present in pACT2. Specificity ofinteraction for with the human androgen receptor (hAR) was determined byexamining the liquid LacZ activity of cDNA clones in the presence of theGAL4 DBD:hAR fusion protein versus the activity observed in the presenceof the GAL4 DBD alone. After sequencing and examination of the GenBankdatabase, it was determined that one interacting clone was encoded byARCAP.

The yeast two-hybrid system was also actively used to confirm theinteraction of ARCAP with hAR. pAS2-constructs were co-transformed withpACT2-ARCAP or pACT2 alone into yeast host Y1187 according to themanufacturer's protocol (Clontech). Trp-positive, Leu-positive colonieswere inoculated in triplicate onto selective media and grown at 30° C.overnight in the presence or absence of 1 μM DHT. Samples were dilutedto an A₆₀₀ of 0.2 and re-grown to an A₆₀₀ of 0.6–0.8. Samples weredivided into three aliquots of 1 ml each. Cells were recovered bycentrifugation at 14,000 rpm for 5 minutes, washed once with buffer Z(0.1 M sodium phosphate, pH 7.0, 10 mM KCl, and 10 mM MgSO₄), andresuspended in 800 μl of buffer Z containing 21 μl of 2-mercaptoethanol.Then 10 μl of 0.1% SDS were added, followed by the addition of 50 μl ofchloroform. Samples were vortexed for 1 minute and incubated at 30° C.,and then 200 μl of o-nitrophenyl-β-D-galactopyranoside (4 mg/ml inbuffer Z) were added. Reactions were timed and terminated upon observingan obvious yellow color or after 1 hour by the addition of 500 μl of 1 MNa₂CO₃. A₄₂₀ of the samples was determined, and activity was calculatedas follows: (A₄₂₀×1000)/(A₆₀₀×time). All assays were performed intriplicate and repeated at least three times.

Results

The full length ARCAP cDNA and the protein it encodes is describedabove.

ARCAP mRNA levels were assessed in normal human tissues and in variouscell lines, including hepatoma cell lines, using Northern blotting.ARCAP was detected only in normal human heart and skeletal muscletissues. High levels of ARCAP expression was detected in hepatoma cellsincluding 3B, 22T, Huh 7, G2, and A2.

Paired liver tumor and normal tissue adjacent to the tumor were isolatedfrom 40 liver cancer patients. Using Northern blotting, it wasdiscovered that ARCAP mRNA was generally highly expressed in the tumorbut expressed very little, if at all, in the adjacent normal livertissue. Using ARCAP-specific antibodies, the presence of ARCAP proteinin tumor tissue and the general absence of ARCAP protein in the adjacentnormal tissue was confirmed. In situ hybridization of ARCAP mRNA inhuman hepatoma tissues also indicated that ARCAP mRNA was abundant intumor cells but rare in normal cells. These data concerning theexpression profile of ARCAP mRNA and protein indicated that ARCAPexpression is a marker for liver cancer.

To determine cellular localization of ARCAP protein, the ARCAP codingregion was fused to GFP in an expression vector, and the vectortransfected into human hepatoma A2 cells. The fusion protein waslocalized in the nucleus, indicating that ARCAP is a nuclear protein.

A yeast two hybrid system was used to determine whether ARCAP binds toandrogen receptor. A full length androgen receptor was cloned adjacentto a GAL4 DNA binding domain and used as a bait to screen forHis3-positives clones from the human matchmaker liver library(Clontech). The interaction of AR and ARCAP in vivo was confirmed bythis study. To further confirm that ARCAP binds to the androgenreceptor, ARCAP and the androgen receptor were co-expressed as fusionproteins. Since immunoprecipitation of androgen receptor led toisolation of ARCAP and immunoprecipitation of ARCAP led to isolation ofandrogen receptor, the physical interaction of ARCAP and androgenreceptor was confirmed.

To determine whether the physical association between the androgenreceptor and ARCAP protein was biochemically significant, theα-fetoprotein gene promoter was used in a luciferase reporter construct.The α-fetoprotein (AFP) gene is a model system for the studyingdevelopmental control of gene expression. AFP is expressed at a highlevel in fetal liver, but its transcription declines rapidly after birthand is hardly detectable in adult life. However, the AFP gene is oftenreactivated to a high level when hepatomas or teratomas develop (Shulmanet al. (1995) Proc. Natl. Acad. Sci. USA 92:8288–8292). An enhancerregion in the AFP promoter contains an androgen responsive element(ARE). A control isogenic luciferase reporter containing a mutated AREwas also used in the study to determine whether the ARE was responsiblefor mediating any biochemical effects.

G2 cells were transfected with the wild type or control reporter and (1)mock DNA, (2) DNA encoding androgen receptor, (3) DNA encoding ARCAP, or(4) DNA encoding androgen receptor and DNA encoding ARCAP. Little changein luciferase activity was observed when the control reporter was used.However, using the wild type reporter, luciferase activity was increasedat least 2× (relative to the control reporter) when androgen receptorwas expressed. Surprisingly, the simultaneous expression of androgenreceptor and ARCAP resulted in about a 3–4 fold increase in luciferaseactivity, relative to the control reporter containing the mutated ARE.This result indicated that ARCAP augments the transactivation activityof androgen receptor on the AFP promoter.

The above AFP reporter experiments were performed in the presence oftestosterone. To confirm that this effect was dependent on formation ofthe testosterone/androgen receptor complex, the wild type reporter wasco-transfected with (1) mock DNA, (2) DNA encoding androgen receptor,(3) DNA encoding ARCAP, or (4) DNA encoding androgen receptor and DNAencoding ARCAP, in the presence or absence of testosterone. The resultsclearly indicated that the enhanced transactivation activity forandrogen receptor due to ARCAP was dependent on testosterone.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the invention, whichis defined by the scope of the appended claims. Other aspects,advantages, and modifications are within the scope of this invention.

1. A substantially pure polypeptide comprising the amino acid sequenceof SEQ ID NO:2.
 2. A method of screening for a compound that decreasesandrogen receptor-mediated transactivation, the method comprisingcontacting the polypeptide of claim 1 with a protein complex includingan androgen receptor, in the presence of a candidate compound; measuringthe extent of binding between the polypeptide and the protein complex;and determining whether the extent of binding is less than the extent ofbinding between the polypeptide and the protein complex in the absenceof the candidate compound, wherein an extent of binding in the presenceof the compound less than the extent of binding in the absence of thecompound indicates that the candidate compound decreases androgenreceptor-mediated transactivation.
 3. The method of claim 2, wherein thecandidate compound is a peptide, a peptidomimetic, a peptoid, or a smallmolecule.
 4. The method of claim 3, wherein the peptide is anoligopeptide or a polypeptide.
 5. The method of claim 3, wherein thepolypeptide and the protein complex are in a cultured cell.
 6. Themethod of claim 5, wherein the cell is a cultured mammalian cell.
 7. Themethod of claim 6, wherein the cultured cell is a human cell.
 8. Themethod of claim 7, wherein the candidate compound is a peptide, apeptidomimetic, a peptoid, or a small molecule.
 9. The method of claim8, wherein the peptide is an oligopeptide or a polypeptide.